Theradiagnostics for cancer Patrick Willems GENDIA Antwerp, Belgium

Theradiagnostics

for cancer

Patrick Willems

GENDIA

Antwerp, Belgium

Treatment of cancer

• Surgery

• Radiation

• Chemotherapy

• Personalised treatment

Personalized cancer treatment

Immunotherapy

to modulate immune response :– Interferon (IFN) alfa-2b, IL2 (interleukin 2) – CTLA-4 inhibitors– PD-1 inhibitors – PD-L1 inhibitors

Targeted therapy with designer drugs

to target the genetic cause of the tumor– EGFR inhibitors– BRAF inhibitor – MEK inhibitor

Bottlenecks in personalized cancer treatment

• Immunotherapy

Extremely expensive (100-300.000 Euro/year)

Few biomarkers (companion diagnostics)

• Targeted therapy with designer drugs Very expensive (50-100.000 Euro/year)

Biomarkers (companion diagnostics)

Bottlenecks in personalized cancer treatment

The very high cost of personalised treatment makes

companion diagnostics (cancer biomarkers) necessary

These are referred to as Theradiagnostics

Theradiagnostics

Tumor DNA (FFPE - biopsy)

Circulating tumor DNA (ctDNA in liquid biopsy)

Market for theradiagnostics

TARGETS DRUGS SEQUENCING

Theradiagnostics market :

40 Billion USD per year

PHYSICIAN

Current paradigm

sampleResult

Pathological studies

PATIENT

PATHOLOGIST

general

treatmentvisit

Lab

PHYSICIAN

Future paradigm

sampleResult

Molecular testing

PHARMA

PATIENT

LAB

Personalised

treatmentvisit

Pathologist

The changing face of cancer diagnosis

Cancer Morbidity and Mortality

New cancers per year in Belgium11 million inhabitants

• Lung : 7.100

• Colon : 6.500

• Prostate : 8.800

• Breast : 9.700

TOTAAL : 65.000

Treatment of cancer

• Surgery

• Radiation

• Chemotherapy

• Personalised treatment :

– Immunotherapy

– Targeted therapy with designer drugs

Immunotherapy for cancer

• CTLA-4 (cytotoxic T-lymphocyte–associated antigen 4) :

ipilimumab, tremelimumab

• PD-1 (programmed death-1) :

nivolumab, pembrolizumab, Lambrolizumab, pidilizumab

• PD-L1 (programmed death-1 ligand) :

BMS-935559, MEDI4736, MPDL3280A and MSB0010718C

• Other checkpoints : TIM3, LAG3, VISTA, KIR, OX40, CD40, CD137

Inhibition immune checkpoints

Biomarkers for immunotherapy for Colorectal cancer

Few biomarkers for immunotherapy

First real biomarker : MicroSatellite Instability (MSI)

Response to pembrolizumab (PD-1 inhibitor) in CRC

MMR-proficient : 0 %

MMR-deficient : 40 %

NEJM : May 30, 2015 (Vogelstein group)

MSI as biomarker for immunotherapy

MMR deficiency

Genomic instability

Large mutation load in CRC

Many mutant proteins – neo antigens

Immune response with immunotherapy

MSI as biomarker for immunotherapy in CRC

MMR deficiency

Genomic instability

Large mutation load in CRC

Many mutant proteins – neo antigens

Immune response with immunotherapy

Treatment of cancer

• Surgery

• Radiation

• Chemotherapy

• Personalised treatment :

– Immunotherapy

– Targeted therapy with designer drugs

Targeted treatment for cancer

Personalised targeted treatment inhibits

specific mutations that cause cancer

These mutations are patient-specific

Mutations can be detected by molecular studies of :

. tumor material (biopsy) : FFPE, fresh or frozen

. blood (liquid biopsy)

Therapy is dependent upon the specific mutation

Personalised medicine

Which genetic anomalies cause cancer ?

Genetics of cancer

• Breast Cancer : 10 %

• Colon cancer : 3-5%

• Prostate cancer : low

• Lung cancer : very low

Majority of cancers are caused by genetic anomalies in the tumor

(somatic mutations)

Minority of cancers is inherited (germline mutations)

Cancer gene mutations

Germline Somatic

Genes Wellknown genes

Limited number of genes

Cancer-specific genes

Still largely unknown

Large number of genes

Cancer-specific (APC) and aspecific (BRAF, EGFR, KRAS) genes

Mutations 1 or 2 mutations per patient

Large number of different mutations

Unknown (novel-private) mutations

Inactivating sequence mutations

Many mutations in each patient

Limited number

Recurrent mutations

Few inactivating sequence mutationsInactivating hypermethylation (MLH1)Activating sequence variantsAmplification of genes (HER2)Deletions of genes (PTEN)

Two step cancer theory (Knudson)

Retinoblastoma (RB1 gene)

Mesothelioma

Uveal melanoma (BAP1 gene)

Two step cancer theory (Knudson)

Inherited cancer :1. Germline mutation in all cells

2. Somatic mutation in cancer cell

Sporadic cancer :1. No germline mutation

2. Somatic mutations in the 2 gene copies in cancer cell

Multistep cancer theory (Vogelstein)

Vogelstein et al, Science Aug 22, 2013

Cancer genes and mutations

• 140 driver genes • 60 % Tumor suppressor genes• 40 % Oncogenes

• > 1000 driver gene mutations(Most tumors 2-10 driver gene mutations)

• Millions of passenger gene mutations(Most tumors 10-100 passenger gene mutations)

Mutations in cancer

• Gate keeper mutations : transforms normal cell into tumor cell

Rb in retinoblastoma

APC in colon cancer

• Driver mutations : confers growth advantage to tumor cell

HER2 in breast cancer

KRAS in colon cancer

• Passenger mutations : accidental mutation not conferring

growth advantage to tumor cell

Any gene

Also driver gene

Driver and passenger gene mutations

Tumors with high mutation load

due to Mutagens or genomic instability

form many neoantigens

and are candidates for immunotherapy

TUMOR MUTATIONS EXPLANATION

HNPCC 1782 Genomic instability

Lung 150 Mutagen (smoke)

Melanoma 80 Mutagen (sun)

Somatic mutations

P

GENE MECHANISM TARGETED THERAPY

APC Inactivating mutation -----------

TP53 Inactivating mutation -----------

EGFR Activating point mutations Gene Amplification Overexpression ligands Overexpression nuclear EGFR

Cetuximab, panitumumaberlotinib, gefitinib, afatinib

KRAS Activating point mutations Tipifarnib, lonafarnib

BRAF Activating point mutations Dabrafenib, sorafenib,vemurafenib,

NRAS Activating point mutations MEK162

PIK3CA Activating point mutations

Inactivating somatic mutations in cancer

P

Breast Lung Colon Prostate

Cancer-specific gene

BRCA --- APCMLH1

---

TP53 23 34 48 16

Activating somatic mutations in cancer

P

Melanoma Breast Lung CRC Prostate

KRAS 17 35 5

NRAS 13-25 3-5

BRAF 10-50 1-4 8-15

PIK3CA 26 4 22 2

EGFR 34 ?? 4

CTNNB1 2-3 48 4

Cell growth and survival pathway

Cell growth pathway

• Ligands

• Receptors : EGFR

• Secondary messengers : 2 pathways :

1. MAPK / RAS pathway : RAS, BRAF, MEK, ERK, Cyclins, CDK4/6

2. mTOR / AKT pathway : PIK3CA, PTEN, AKT, mTOR

Classical treatment in colon cancer

• Surgery

• Chemotherapy

• In case of EGFR mutation or overexpression

Start anti EGFR therapy :

• mAB : cetuximab, panitumumab

• TKI : erlotinib, gefitinib, afatinib

EGFR mutations

• Lung Ca : activating mutations in TK domain

• Glioblastoma : activating mutations in Extracellular domain

• Colorectal ca : unclear :

Overexpression membrane EGFR (mEGFR)

Overexpression nuclear EGFR (nEGFR)

Gene Amplification

Overexpression ligands

Activating point mutations

EGFR status

Anti-EGFR therapy

mAB : cetuximab, panitumumab

TKI : erlotinib, gefitinib, afatinib

EGFR Resistance : T790M mutation

Inhibitors of EGFR with the T790M mutation :

AZD9291

CO-1831

EGFR resistance : KRAS and BRAF mutations

EGFR

KRAS

WILD

TREATMENT RELAPSE

EGFR resistance in CRC: KRAS and BRAF mutations

Resistance against EGFR therapy

– KRAS mutation : 40 %

– BRAF mutation : 8-15 %

– NRAS mutation : 1-6 %

• Mostly pre-existent – selection due to anti-EGFR treatment

• Also new due to ongoing mutagenesis ?

Addition of BRAF or MEK inhibitor

EGFR resistance in CRC : PIK3CA mutation

Resistance against EGFR therapy

PIK3CA mutation : 10-30 %

PTEN loss

Addition of mTOR inhibitor

PIK3CA

• PIK3CA encodes p110 subunit of Phosphatidylinositol 3-kinase

PIK3 phosphorylates PI

PI is central in AKT/mTOR pathway

• PIK3CA driver mutations in :– Breast cancer (26 %)– Endometrium (23 %)– Colon (22 %)– Non-tumor : somatic overgrowth syndromes

(Cowden and Clove syndrome)

• Therapy : PIK3, AKT, mTOR inhibitors

Why genetic studies on tumor DNA ?

• Initial diagnosis and prognosis

• Monitoring recurrence – metastasis

Genetic studies in cancer

• Blood DNA

If CRC occurs in different family members :

Genetic studies on DNA from blood to identify a germline mutation (BRCA)

• Tumor

• MSI : in order to determine sensitivity for immunotherapy

• Mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA

to determine sensitivity for targeted therapy

• Liquid biopsy

• Initial theradiagnostics if tumor material is unavailable

• Follow up during cancer treatment

• Screening of high risk patients (HNPCC carriers, BRCA carriers)

Genetic studies of somatic mutations

• DNA studies on tumor material

Analysis of DNA from tumor (FFPE, fresh, frozen)

• Circulating tumor DNA (ctDNA) in Liquid biopsy

Analysis of circulating tumor DNA (ctDNA) in blood

Circulating tumor DNA (ctDNA)

ctDNA

ctDNA from tumor tissue is released through secretion, necrosis and apoptosis,

but mainly through apoptosis.

cell-free DNA (cfDNA) testing

• Cell-free DNA (cfDNA) in plasma of healthy individuals : Mandel and Métais (1948)

• A proportion of cfDNA in pregnant women is fetus-derived (cffDNA) : Lo et al. (1997)

• Non-Invasive Prenatal testing (NIPT) : 2012 : start

2015 : > 1 million tests

  Market : 4 billion USD

• Increased concentrations of cfDNA in the circulation of cancer patients : Leon et al. (1977)

• A proportion of cfDNA is tumor-derived : Stroun et al. (1987)

• Circulating tumor DNA (ctDNA) testing (liquid biopsy) : 2015 : start

  Market : 40 billion USD

Advantages liquid biopsies

• No tissue biopsy needed

• No FFPE fixation

• Profiling the overall genotype of cancer

• primary cancer

• circulating cells

• metastases

• Better evaluation of :

• reaction to therapy

• development of resistance

Tissue biopsy

EGFR

KRAS

WILD

EGFR TREATMENT RELAPSE

TISSUE BIOPSY

Liquid biopsy

EGFR

KRAS

BRAF

WILD

TREATMENT

LIQUID BIOPSY

Companies focusing on Theradiagnostics

• Cynvenio• BGI• Agena Bioscience • Boreal Genomics • Chronix Biomedical • Genomic Health • Guardant Health• Inivata• Molecular MD • Pangaea Biotech• Myriad Genetics• Pathway Genomics• Natera • Personal Genome Diagnostics• Sysmex Inostics• Trovagene • ETC

Theradiagnostics market :

40 Billion USD per year

ct DNA testing on liquid biopsy for CRC

1. DESCRIPTION : ct DNA testing on liquid biopsies :

90 mutations in 9 cancer genes :

• EGFR• TP53• KRAS• BRAF• PIK3CA

2. SAMPLE : blood in specific test kits with Streck tubes (GENDIA)

3. TURNAROUND TIME : 3 weeks

4. PRICE : 650 Euro

• NRAS• CTNNB1• GNAS• FOXL2

How offer ctDNA testing to your patients ?

1. Take blood yourself :

Email ctDNA@GENDIA.net to ask for kits

2. Refer to our consultation :

Email ctDNA@GENDIA.net to ask for an appointment

www.circulatingtumorDNA.net

www.circulatingtumorDNA.net